Diabetes mellitus is a major health problem in the U.S. and the Type II disease will soon reach epidemic proportions. Type I is due to absolute insulin deficiency, whereas Type II is due to impairment of insulin action. In either case an understanding of the molecular basis of the initial stages of insulin action, receptor binding and tyrosine kinase activation, will be essential for the development of improved agents for the treatment of the disease. However, despite more than 25 years intensive study, this remains elusive. Thus the longterm objective of this proposal is to elucidate the molecular mechanisms underlying the formation of the insulin-receptor complex. The weight of evidence suggests that both insulin and the receptor have two distinct binding sites. This has lead to the formulation of a number of hypothetical models to explain complexities of the insulin-receptor interaction. Studies in this laboratory using systematic, mutational analysis have identified and characterized the functional epitope of one of these receptor binding sites. This strategy will now be used to localize and characterize the functional epitope of the other ligand binding site and also to evaluate proposed hypothetical models of insulin binding. The Specific Aims of the proposed studies are: 1) Identification of receptor domains and sub-domains contributing to the second receptor ligand binding site; 2) Localization of the functional epitope of the second ligand binding site by alanine scanning mutagenesis; 3) Evaluation of the kinetics of insulin and analog binding to alanine mutants of determinants of the functional epitope; 4) Evaluation of the effects of alanine receptor mutations on negative cooperativity of insulin binding; and 5) Comparison of the functional epitopes of the second ligand binding sites of A and B isoforms of the insulin receptor.